Control of Lytic Function by Mitogen-activated Protein Kinase/Extracellular Regulatory Kinase 2 (ERK2) in a Human Natural Killer Cell Line: Identification of Perforin and Granzyme B Mobilization by Functional ERK2

The signal pathways that control effector function in human natural killer (NK) cells are little known. In this study, we have identified the critical role of the mitogen-activated protein kinase (MAPK) pathway in NK lysis of tumor cells, and this pathway may involve the mobilization of granule components in NK cells upon interaction with sensitive tumor target cells. Evidence was provided by biological, biochemical, and gene transfection methods. NK cell binding to tumor cells for 5 min was sufficient to maximally activate MAPK/extracellular signal–regulatory kinase 2 (ERK2), demonstrated by its tyrosine phosphorylation and by its ability to function as an efficient kinase for myelin basic protein. MAPK activation was achieved in NK cells only after contact with NK-sensitive but not NK-resistant target cells. In immunocytochemical studies, cytoplasmic perforin and granzyme B were both maximally redirected towards the tumor contact zone within 5 min of NK cell contact with tumor cells. A specific MAPK pathway inhibitor, PD098059, could block not only MAPK activation but also redistribution of perforin/granzyme B in NK cells, which occur upon target ligation. PD098059 also interfered with NK lysis of tumor cells in a 5-h ⁵¹Cr-release assay, but had no ability to block NK cell proliferation. Transient transfection studies with wild-type and dominant-negative MAPK/ERK2 genes confirmed the importance of MAPK in NK cell lysis. These results document a pivotal role of MAPK in NK effector function, possibly by its control of movement of lytic granules, and clearly define MAPK involvement in a functional pathway unlinked to cell growth or differentiation.     

Validity of establishing pediatric reference intervals based on hospital patient data: A comparison of the modified Hoffmann approach to CALIPER reference intervals obtained in healthy children

Objectives

To compare pediatric reference intervals calculated using hospital-based patient data with those calculated using samples collected from healthy children in the community as part of the CALIPER study.

Methods

Hospital-based data for 13 analytes (calcium, phosphate, iron, ALP, cholesterol, triglycerides, creatinine, direct bilirubin, total bilirubin, ALT, AST, albumin and magnesium), measured on the Vitros 5600, collected between 2007 and 2011 were obtained. The data for each analyte were partitioned by age and gender as previously defined by the CALIPER study. Outliers in each partition were removed using the Tukey method. The cumulative distribution function (cdf) was then determined for each analyte value following which, the inverse cdf values of a standard Gaussian distribution were calculated. The analyte values were plotted against the inverse cdf of the standard Gaussian distribution. Piece-wise regression determined the linear portion of the resulting graph using the statistical software R. Linear regression determined an equation for the linear portion in each partition and reference intervals were calculated by extrapolating to identify the 2.5th and 97.5th centiles in each partition based on the inverse cdf values (which would correspond to the values − 1.96 and 1.96 of the Gaussian distribution). Using the 90% confidence intervals for the reference intervals defined by CALIPER and the Reference Change Value (RCV) as the criteria, these calculated reference intervals were compared to those reported previously by CALIPER. Reference samples were also measured on the Vitros 5600 analyzer in an attempt to validate the calculated reference intervals.

Results

In general, the reference intervals calculated from hospital-based data were generally wider than those calculated by CALIPER. None of the reference intervals calculated using the Hoffmann approach fell completely within the 90% confidence intervals calculated by CALIPER.

Conclusions

These results suggest that calculating pediatric reference intervals from hospital-based data may be useful, as a guide, in some cases but will likely not replace the need to establish reference intervals in healthy pediatric populations.     

Kelp-forest dynamics controlled by substrate complexity

The factors that determine why ecosystems exhibit abrupt shifts in state are of paramount importance for management, conservation, and restoration efforts. Kelp forests are emblematic of such abruptly shifting ecosystems, transitioning from kelp-dominated to urchin-dominated states around the world with increasing frequency, yet the underlying processes and mechanisms that control their dynamics remain unclear. Here, we analyze four decades of data from biannual monitoring around San Nicolas Island, CA, to show that substrate complexity controls both the number of possible (alternative) states and the velocity with which shifts between states occur. The superposition of community dynamics with reconstructions of system stability landscapes reveals that shifts between alternative states at low-complexity sites reflect abrupt, high-velocity events initiated by pulse perturbations that rapidly propel species across dynamically unstable state–space. In contrast, high-complexity sites exhibit a single state of resilient kelp–urchin coexistence. Our analyses suggest that substrate complexity influences both top-down and bottom-up regulatory processes in kelp forests, highlight its influence on kelp-forest stability at both large (island-wide) and small (<10 m) spatial scales, and could be valuable for holistic kelp-forest management.

Kelp-forest ecosystems exhibit rich and varied spatiotemporal dynamics. Prominent among these are dramatic shifts between kelp-dominated forests and so-called urchin barrens from which macroalgae are almost entirely absent due to intense urchin grazing (1, 2). Phase shifts between kelp and barren states have long been associated with structural changes to kelp-forest communities, such as the addition or removal of sea-urchin predators (3, 4) or changes in the environment such as shifting water temperatures (4–7). Kelp forests are also subject to stochastic perturbations such as large wave, marine disease, and anomalous warm water events that might perturb kelp forests between alternative stable states (8, 9). However, distinguishing phase shifts and alternative stable states is a major challenge (10). This is partially because both slow environmental change and relatively rapid stochastic perturbations often appear to act synergistically and with episodic urchin recruitment events that, due to their large regional extent, decouple rates of urchin grazing from the local density-dependent regulation of their populations (11, 12).Although consensus is emerging that the maintenance of kelp-dominated forests is driven by a combination of top-down and bottom-up processes, the mechanisms underlying these processes—and hence the optimal means to control and avoid tipping points to the urchin-barren state—appear varied and often unclear (1, 13). For example, top-down processes contributing to kelp-forest stability include the effects of predators and disease on urchin grazing behavior and mortality rates (14–18), emphasizing the need for management strategies that preserve or restore top-down forms of urchin control (19, 20). On the other hand, bottom-up processes affecting kelp growth and senescence rates, and the retention of drift algae that urchins prefer to consume, are also known to contribute to kelp-forest stability, emphasizing management strategies that differ from those of direct urchin control (21–25). We hypothesize that substrate complexity modifies both top-down and bottom-up processes structuring urchin–kelp interactions, e.g., provisioning habitat for urchin predators and increasing the retention of drift algae for urchins.Here we apply the perspective of stochastic dynamical systems to the study of kelp forests not to determine the specific mechanisms or feedbacks that underlie kelp-forest dynamics but rather to infer an environmental variable that influences their relative strength and net expression. The dynamical-systems perspective conceptualizes a system’s community states and dynamics using the ball-in-cup heuristic of stability and resilience (26, 27), formally described by a (quasi-)potential stability landscape (28, 29). A system with alternative stable states exhibits a multimodal landscape with two or more basins of attraction (cups) over which it travels in time due to endogenous drivers (e.g., species interactions) and external perturbations. Because most perturbations are directionally random and small, communities spend more time in states at the bottom of the attracting basins than they do on their slopes and cusps, with deeper and steeper-sloped basins corresponding to more stable and resilient community states whose dynamics are dominated by negative feedbacks (28). Previous work has utilized this characteristic of stochastic dynamical systems to make use of large-scale spatial variation in community structure to infer what biotic and environmental conditions may alter the stability of various ecological systems, including tropical and temperate forests and desert biomes (4, 30–32). For example, Scheffer et al. (33) used satellite-derived spatial variation in the frequency distributions of percentage of tree cover values to infer that boreal biomes exhibit between one and three different alternative stable states whose number and nature depend on mean July temperature, where empirical system–state frequency histograms represent negative potential (i.e., a mirror image of a ball-in-cup stability landscape reflected across the x axis). Similarly, Ling et al. (4) combined spatial survey data with translocation experiments to infer bistability in response to urchin densities in Tasmanian kelp forests. The approach underlying these inferences has been referred to as potential analysis (34).Using spatially fixed and replicated long-term time series of kelp-forest community dynamics around San Nicolas Island, CA, we extended the application of potential analysis to include the temporal domain to more rigorously infer their condition-dependent stability landscapes and shifts in community structure. Our analyses reveal kelp-forest communities around San Nicolas Island to exhibit dramatic, perturbation-induced shifts between kelp-dominated forests and urchin-barren states only when the complexity of the underlying substrate is low and that similarly perturbed high-complexity substrates permit only a single persistent state of resilient kelp–urchin coexistence. We infer that substrate complexity at San Nicolas Island controls the relative strength of the many negative and positive feedbacks that have been described in kelp forests and that a greater understanding of its influences is likely to increase the effectiveness of management efforts seeking to conserve and restore their existence.     

Outbreak of invasive meningococcal disease caused by a meningococcus serogroup B in a nursery school,Wallonia, Belgium, 2018

Although most invasive meningococcal disease (IMD) cases are sporadic without identified transmission links, outbreaks can occur. We report three cases caused by meningococcus B (MenB) at a Belgian nursery school over 9 months. The first two cases of IMD occurred in spring and summer 2018 in healthy children (aged 3–5 years) attending the same classroom. Chemoprophylaxis was given to close contacts of both cases following regional guidelines. The third case, a healthy child of similar age in the same class as a sibling of one case, developed disease in late 2018. Microbiological analyses revealed MenB with identical finetype clonal complex 269 for Case 1 and 3 (unavailable for Case 2). Antimicrobial susceptibility testing revealed no antibiotic resistance. Following Case 3, after multidisciplinary discussion, chemoprophylaxis and 4CMenB (Bexsero) vaccination were offered to close contacts. In the 12-month follow-up of Case 3, no additional cases were reported by the school. IMD outbreaks are difficult to manage and generate public anxiety, particularly in the case of an ongoing cluster, despite contact tracing and management. This outbreak resulted in the addition of MenB vaccination to close contacts in Wallonian regional guidelines, highlighting the potential need and added value of vaccination in outbreak management.     

Utility of selection methods for specialist medical training: A BEME (best evidence medical education) systematic review: BEME guide no. 45

Background: Selection into specialty training is a high-stakes and resource-intensive process. While substantial literature exists on selection into medical schools, and there are individual studies in postgraduate settings, there seems to be paucity of evidence concerning selection systems and the utility of selection tools in postgraduate training environments.

Aim: To explore, analyze and synthesize the evidence related to selection into postgraduate medical specialty training.

Method: Core bibliographic databases including PubMed; Ovid Medline; Embase, CINAHL; ERIC and PsycINFO were searched, and a total of 2640 abstracts were retrieved. After removing duplicates and screening against the inclusion criteria, 202 full papers were coded, of which 116 were included.

Results: Gaps in underlying selection frameworks were illuminated. Frameworks defined by locally derived selection criteria, and heavily weighed on academic parameters seem to be giving way to the evidencing of competency-based selection approaches in some settings.

Regarding selection tools, we found favorable psychometric evidence for multiple mini-interviews, situational judgment tests and clinical problem-solving tests, although the bulk of evidence was mostly limited to the United Kingdom. The evidence around the robustness of curriculum vitae, letters of recommendation and personal statements was equivocal. The findings on the predictors of past performance were limited to academic criteria with paucity of long-term evaluations. The evidence around nonacademic criteria was inadequate to make an informed judgment.

Conclusions: While much has been gained in understanding the utility of individual selection methods, though the evidence around many of them is equivocal, the underlying theoretical and conceptual frameworks for designing holistic and equitable selection systems are yet to be developed.